This project follows our discovery of ganglioside presence in the nuclear envelope (NE) of neural cells, and the functional association of GMtwith a Na-Ca exchanger in the same membrane. In potentiating the exchanger activity, GM1 was shown to bind to this protein with unusually high affinity, a phenomenon that was not shared by these molecules in the plasma membrane. One goal of the proposed studies is to elucidate the molecular basis of this unusual association by determining which of the several known exchanger isoforms has the ability to (a) traffic to the NE and (b) bind GM1. Various isoforms of the exchanger will be transfected into Jurkat cells, which we have found do not normally express the GM1-exchanger complex in the nucleus, and the NE examined for evidence of such expression. Exchanger isoforms having this ability will be compared for common structural motifs that appear to promote high affinity binding (likely to include basic amino acids);the structures will be altered through site-directed mutagenesis to determine whether such exchangers, following transfection, have lost the ability to sequester GM1 in the NE. We will test the hypothesis that the GM1-exchanger complex occurs in the NE in association with ganglioside GD1a and neuraminidase, both of which were also shown to occur in the NE. The presence of these 2 molecules could constitute a supply mechanism to ensure sufficient GM1 for potentiating the exchanger. The functional role of the GM1-exchanger complex, postulated to remove elevated nuclear calcium, will be tested by comparing nuclear calcium changes in cells which do and do not have the GM1-exchanger complex. The functional role will also be tested in vivo with knockout mice lacking GM1, which were found to be highly susceptible to kainate-induced seizures. Demonstrated attenuation of such seizures by administered LIGA-20, a membrane-permeant derivative of GM1, will be further studied by determining the role of nuclear GM1/exchanger in modulating the seizure mechanism.